Several disorders in reproductive medicine are results of changes in smooth muscle contractile activity. Increased myometrial contraction can lead to preterm labor, which affects 12% of the US population. However, the mechanisms involved in the transition from uterine quiescence to contractility at the onset of labor are not well-known. As a result, the management of labor disorders such as preterm birth is poor, particularly tocolytic therapies, which could delay preterm labor, but have not been effectively proven and tested for this purpose. The slow progress in understanding myometrial contractility and tocolytic management of preterm labor can be attributed to the lack of faithful in vitro models of the myometrium, as well as the ability to efficiently screen tocolytic compounds in a high-throughput fashion. While in vivo models are used in the uterine contractility research, pronounced differences in how animals and humans give labor mean that pathological changes have different biological bases and responses to drugs. Beyond these intrinsic differences, animal models are time-consuming, costly, and ethically challenging. Alternatively, ex vivo human myometrial tissue are useful models for uterine contractility research. Yet, ex vivo models are not ideal for robust studies on uterine contractility research, as they suffer from ethical issues, sample inconsistencies, and scarcity. As a result, in vitro assays have been explored as cheaper and robust alternatives to study the efficacy of tocolytic substances to predict efficacy in humans, or as a compound screen before in vivo testing. The development of in vitro cell culture models and organ systems has greatly facilitated the study of gene expression and pathway regulation within human myometrial tissues, as well as identify and characterize target pathways. The success of these studies confirms the ability to study uterine contractility at the cellular level by examination of electrical conduction and protein expression. However, in general, in vitro cell culture models are limited by their accuracy, likely due to the fact that most in vitro testing is performed on two-dimensional (2D) glass or plastic surfaces, or organ bath systems that do not fully represent the native human myometrium environment. Specifically to uterine contractility research, while in vitro models allow for a myriad of metrics related to uterine activity to be monitored in a controlled environment, these models are prone to spontaneous activity involving both activating and spontaneous contractile mechanisms that require suppression of the activating mechanism to achieve consistency in results. Additionally, limitations still remain in 2D cell culture and in vitro uterine tissue models in studying tissue-lvel physiology and cellular pathways, respectively, resulting in data lacking context, detail, accuracy and mostly reproducibility. Given these limitations, this proposal looks toward three-dimensional (3D) models, which more accurately can represent the native tissue environment. Specifically, a recently explored assay, the BiO Assay, will be applied to contractility research as the C-BiO Assay. The basis of the C-BiO Assay is magnetic printing of cells. Cells are incubated with nontoxic magnetic nanoparticles that render the cells magnetic. Using ring-shaped magnets, the cells are then printed in 96-well plates into 3D rings, which close over time and at a rate that varies with compound concentration. The C-BiO Assay uses label-free metrics, so it does not require any reagents, dyes, or specialized equipment. Furthermore, data is gathered using a mobile device, which can be programmed to image whole plates at specific time points, avoiding the time-consuming imaging of individual wells under a microscope or reading plates on a plate reader, that is involved in 2D in vitro assays. Our hypothesis is that the C-BiO Assay will apply the benefits of 3D cell culture to an area of need, the lack of a faithful in vitro myometrial mode for contractility research, while being faster than other assay systems. In this Phase I proposal, the parameters of the C-BiO Assay will be optimized for high-throughput screening. Then, the assay will be compared to other 2D and 3D assays, and validated as a measure of smooth muscle contraction. This assay will integrate: Capability to rapidly print 3D cell cultures with relevant extracellular matrix Real-time and label-free quantification of ring closure, which correlates with cell function Ability to investigate the basal cytotoxicity of particular compouns and their mechanisms of actions Tools for high-throughput analysis that could significantly cut the time and cost of data collection The end result is an assay that mimics the myometrial structure and physiology, particularly smooth muscle contraction, and allows for high-throughput testing to efficiently screen tocolytic compounds for efficacy and toxicity. Aims Aim 1 - Optimization of the Magnetic Levitation and Printing of Myometrial 3D Cell Cultures for the C-BiO Assay Aim 2 - Validation of the 3D Myometrial C-BiO Assay.